1. Field of the Invention
This invention relates to the development of a novel composition having surface coating properties useful in immunoassay procedures. More particularly this invention relates to development of a kit combining reagents and a method to produce immunochemical devices having highly defined reactive surface properties. The system employs a formulation of chitosan and an organic acid to coat plastic microtiter wells. The chitosan is fixed with an alkaline solution to produce a thin film insolubilized on the plastic. Concomitant with fixation, mild oxidation of the surface coating produces highly reactive groups that will bind antigen or antibody to the wells of the microtiter plate.
2. Background Information
The monitoring of medically important substances and foreign substances in the environment has become increasingly dependent on immunoassay technology. Antibody directed against a molecule has the ability to act as a reagent for the estimation of its target molecule in a fluid sample. Immunoassay technology is a widely practiced art. In general the assay requires immobilization of either antibody or antigen to an insoluble surface (substrate). The reaction proceeds with the addition of reagents, and the formation of an antibody/antigen complex. Antibody binding can then be measured by colorimetric, radiometric, turbidometric, and fluorometric means.
A variety of immunochemical substrates exist including materials such as metal, cellulose, glass, and plastic. Substrates may take the form of tubes, beads, filters, dipsticks, and particles. The most widely practiced art is the microtiter well formed in plastic. These devices are formed of any plastic including polystyrene, polycarbonate, polypropylene, or polyvinyl chloride. Typically these devices are in strips of either eight or twelve wells ganged together, or in plates of up to 96 wells. The plastic may act as the substrate, or the microtiter well manufactured with an added ingredient to increase the binding of agents to the surface. An example is found in U.S. Pat. No. 4,657,873 (Gadero, 1987) which describes activating plastic surfaces with a phenyl-lysine copolymer to provide a surface for subsequent crosslinking reactions. The consistency and uniformity of these coatings directly impact the performance of the test.
A variety of strategies to further increase the reactivity of microtiter wells are available to practioners of this art. These include coatings of bovine serum albumin, poly-1-lysine, polyacrylamide, alginate, carrageenan, and others. See, p. Tijssen, PRACTICE AND THEORY OF ENZYME IMMUNOASSAYS, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (1985). The goal of coating solutions in immunoassay test procedures is to increase and control the binding of reactants. Desired traits of solid phase coatings include: high ligand capacity, diversity of immunoreactants, minimal dissociation, negligible denaturation of antigen or antibody, controlled orientation, inexpensive, uniform, controlled spacing of epitopes and paratopes, minimal interaction between coating and secondary regents, no binding effects (background) or steric hindrance, immunochemically transparent, and negligible optical interference. Bovine serum albumin is the most widely employed reagent in coating technology. Absoption of albumin to plastic microtiter plates varies considerably between types of plastics. There is considerable lot to lot variation of both microtiter plates and albumin. The pH of the binding solution is also a factor that results in decreased absorption. Considerable assay to assay variability will occur from the use of albumin coatings. Additionally, the small number of amine bearing lysine residues limits subsequent coupling via bridging molecules such as glutaraldehyde.
Poly-1-lysine offers greater sensitivity for coupling reactions, however considerable non-specific binding occurs. Membranes and particles of chitosan, a copolymer of glucosamine and galactosamine derived by hydrolysis of chitin, have been used as a support in immunoassay procedures after the material was crosslinked into an insoluble substrate. The poly hexosamine chitosan, a derivative of chitin, has been the subject of numerous reviews (CHITIN, CHITOSAN AND RELATED ENZYMES, 1984, ACADEMIC PRESS INC.). Chitosan has been analyzed using sodium nitrite in a general method for determination of mucopolysaccarides (Tsuji et al., 1969; Clin. Pharm. Bull. 17:1505). Chitosan has been used as an immobilizing agent for enzymes and other biological proteins. U.S. Pat. No. 4,167,447 (Masri et al., 1979) describes a precipitation procedure for insolubilization of chitosan and macromolecules using alkaline conditions. U.S. Pat. No. 4,089,746 (Masri et al., 1978) insolubilized enzymes on chitosan gels using a crosslinking agent. U.S. Pat. No. 4,094,743 (Leuba, 1978) used chitosan flakes and powders as a support with a dialdehyde crosslinker. U.S. Pat. No. 4,760,024 (Lantero, 1988) produced a spherical aggregate of chitosan and enzymes using glutaraldehyde. The technology cited above require crosslinking of the chitosan with glutaraldehyde to stabilize the chitosan and provide reactive areas.
Each of the foregoing technologies meets at least one of the goals for an optimal reactive coating but each also contain features that compromise the technology. Serum albumins are not immunochemically transparent and may result in background staining. Polylysine and its various copolymers result in nonspecific binding interactions. Passive absorption to unmodified plastics results in nonuniformity. Thus, a need continues to exist for a reliable and cost effective material for immobilization.